In the life cycle of HIV, the Rev protein regulates the temporal switch from the early regulatory to the late lytic phase by binding to a highly structured RRE (Rev Responsive Element) RNA. Two questions regarding Rev:RRE RNA interactions still remain unanswered, namely 1) the primacy of sequence or secondary structure of RNA in determining REV binding, and 2) the roles of oligomerization and effector domains of REV protein in RNA binding. We performed real time kinetic studies of REV:RRE interactions by the SPR biosensor technique. For this purpose, we developed a novel method of immobilizing RNA on the sensor surface that may also be of general use for detecting novel nucleic acid binding proteins. To more precisely locate the RNA binding domain(s) on Rev, a series of REV peptides representing the minimal REV motifs were evaluated for RNA binding by SPR. A minimal sequence between the 23rd and 87th residues comprising the RNA binding, NLS, oligomerization and effector motifs of REV exhibited in vitro RNA binding kinetics reminiscent of REV. We have further characterized the biochemical properties of the cellular ds RNA binding protein, TRBP that bound to the RRE RNA. TRBP was a potent inhibitor of the interferon induced PKR kinase in vitro and in vivo. Both PKR and TRBP were capable of dimerizing and the PKR inhibition by TRBP was mediated by heterodimerization with PKR. We have extended our studies on the effect of HIV-1 NEF protein on T lymphocyte CD4 receptor. We demonstrated that Nef downregulates CD4 expression through a bi-modal mechanism through endocytosis of cell surface receptor and repression of biosynthesis and transport of nascent CD4. Using the yeast two hybrid system of genetic screening, we identified two HeLa cell proteins that interact with Nef (Nip-29 and NIP-50). NIP 29 is a partial cDNA clone encoding a polypeptide of 255 amino acids. The polypeptide was rich in basic amino acids and the C-terminal 60 amino-acids appear to function as a trans-activator in yeast and this domain also appears to be critical for Nef binding. When a HA epitope tagged NIP-29 was transfected into HeLa cells, it was found to be exclusively nuclear in location with nucleolar concentration. NIP-29 does not show any significant homology with sequences currently in the data banks but the C-terminal end shows homologies with transcription factors suggesting that it is itself a transcription factor. We have obtained strong evidence to suggest that Nef may interact in vitro with a novel plasma membrane associated serine/threonine protein kinase that does not appear to belong to the PKA or PKC family and might belong to the PAK kinase family.